Project Summary Notch signaling is a fundamental mechanism through which a majority of multicellular animals regulate cell biology. Mammalian organisms express four Notch receptors that are activated by nearly identical mechanisms. Many years of Notch research has led to a dogma whereby active Notch fragments (NICD molecules) associate with a co- transcription factor complex that in turn binds to DNA as either monomers, or as head- to-head homodimers to drive transcription. There remain however significant gaps in our understanding of Notch. Chief among these gaps are an understanding of how all the Notch receptors are integrated to produce the overall Notch signaling output from a cell. The main goal of this proposal is to address these gaps in knowledge. Our preliminary results have now identified a novel mode of Notch signaling, heterodimerization between different NICD molecules. Moreover, we have also determined that even different Notch NICD homodimers display unique transcriptional activities on various synthetic promoters. We hypothesize that both heterodimerization of NICD molecules and unique promoter preferences of various NICD homodimers cooperate to diversify the Notch signaling mechanism. To test this hypothesis, we propose two aims in which NICD heterodimerization will be characterized based on the fundamental rules of heterodimerization and the promoter binding activity of various NICD homodimers will be compared. Given the critical importance of Notch signaling to cell biology under both healthy and diseased states, these results will add fundamental knowledge that will be valuable in the struggle to improve human health.